Project Summary Alzheimer's disease (AD) is the most common form of dementia, affecting more than 5 million people in the US; about one in eight people that are 65 and older have the disease. AD cannot be prevented, cured or slowed. Recently, GWAS studies revealed that people with an arginine-47-histidine (R47H) mutation of the Triggering receptor expressed on myeloid cells 2 (TREM2) gene are 3-5 times more likely to get AD than those without the mutation. TREM2 is a microglia receptor that detects phospholipids and transmits intracellular signals through an associated adapter DAP12. The TREM2?DAP12 complex promotes activation and proliferation of microglia in response to AD lesions, presumably through recognition of phospholipids exposed on damaged neurons or associated with amyloid-? (A?) plaques. However, the R47H mutation prevents TREM2 binding to phospholipids ligands, which impairs its capacity to sustain microglial responses to AD lesions. Notably, heterozygosity for R47H is sufficient to considerably increase AD risk. Moreover, TREM2 deficiency and haploinsufficiency in the 5XFAD mouse model of AD accelerate disease progression in a dose- dependent fashion. These data suggest that AD progression is quite sensitive to changes in TREM2 expression. Thus, modulation of TREM2 expression levels may offer a powerful strategy for harnessing protective functions of TREM2. Early work on TREM2 reported that IL-4 increases TREM2 surface expression, LPS or IFN? cause loss of expression, while ADAM10 and ADAM17 proteases cleave TREM2 from the microglia cell surface. However, beyond these isolated mechanisms, little is known about regulation of TREM2 surface expression. By performing an unbiased CRISPR-Cas9 knockout library screening for TREM2 surface expression in the microglial cell line BV2, we have identified two novel regulators, DOK1 and TMEM131. DOK1 is a negative regulator of the Ras-ERK pathway. Nothing is known about its function in microglia and brain physiopathology. Our preliminary data demonstrate that DOK1 promotes TREM2 surface expression. TMEM131 is a functionally uncharacterized two-pass transmembrane protein. Almost nothing is known about this molecule. Our preliminary data show that TMEM131 inhibits TREM2 surface expression. In in vitro experiments with knockout and overexpressing BV2 lines, we will test the hypothesis that DOK1 and TMEM131 regulate surface expression of TREM2 by forming a complex with TREM2?DAP12 that is efficiently exported from ER and Golgi to the cell surface. By analyzing Dok1?/? and Tmem131?/? mice in the 5XFAD model of AD, we will determine the impact of DOK1 and TMEM131 on microglial responses to A? plaques and A?-related pathology in vivo. Overall, these studies will identify key pathways that regulate TREM2 expression in steady?state and during A? plaque pathology and will allow us and others to pursue novel, better-targeted therapeutics to boost TREM2 expression.